Design and study of artificial nucleases for DNA photocleavage

人工核酸酶是能够在DNA骨架中诱导单链或双链断裂的分子。 在光激活时切割DNA的分子在光动力疗法（PDT）中尤其重要，光动力疗法是一种已被应用于成功治疗某些癌症和年龄相关性黄斑变性的策略。 目前用于PDT的临床药剂遭受三个主要限制：（i）需要氧气来发挥作用，（ii）在对组织最透明的波长处的光的最小吸收，和（iii）不能产生双链断裂。 该提案概述了一项研究计划，该计划致力于通过引入新的药剂来克服这些限制，这些药剂在缺氧条件下用700-900 nm的光激活时引起DNA双链断裂。 第一个提出的DNA光切割剂家族是基于双核Ru（II）络合物，其吸收>700 nm的光。 这个家族的扩展将包括Rh（II）的掺入，以制造在不存在氧的情况下切割DNA的混合多金属系统。 多金属框架将为DNA切割提供两个反应性位点。 15-16个碱基对内的双重反应性将引入双链断裂，这对于细胞机器来说更难以修复。 一个相关的研究流涉及jadomycins，一个家庭的次级代谢产物，诱导DNA链断裂的存在下，铜（II）或光活化。 虽然它们达不到理想的PDT试剂所概述的性质，但jadomycins对革兰氏阳性和革兰氏阴性细菌以及一些耐药癌细胞系表现出细胞毒性活性。该研究计划将确定直接DNA切割是否有助于细胞内Cu（II）浓度下Jadomycins的总体细胞毒性。 此外，将研究雅多霉素的DNA光裂解特性，特别关注Cu（II）活化与光活化的主导机制。 L-洋地黄毒糖，雅多霉素B的结构特征，在DNA裂解中发挥的作用也将被探讨，以更好地了解如何糖基掺入艾滋病在DNA识别。

Artificial nucleases are molecules capable of inducing single- or double-strand breaks in the DNA backbone.  Molecules that cleave DNA  upon activation with light are especially important in photodynamic therapy (PDT), a strategy that has been applied to the successful treatment of certain cancers and age-related macular degeneration.  Current clinical agents for PDT suffer from three major limitations:  (i) requirement for oxygen to function, (ii) minimum absorption of light at wavelengths most transparent to tissue, and (iii) inability to produce double-strand breaks.  This proposal outlines a research plan that is devoted to overcoming such limitations by introducing new  agents that elicit double-strand breaks in DNA under hypoxic conditions when activated with 700-900 nm light.  The first proposed family of DNA photocleavers is based on dinuclear Ru(II) complexes that absorb light >700 nm.  Extension of this family will include incorporation of Rh(II) to make hybrid multimetallic systems that cleave DNA in the absence of oxygen.  The multimetallic framework will provide two sites of reactivity for DNA cleavage.  Dual reactivity within 15-16 base pairs will introduce double-strand breaks which are more difficult for the cellular machinery to repair.       A related research stream involves  the jadomycins, a family of secondary metabolites that induces DNA strand scissions in the presence of Cu(II) or with light activation.  While  they fall short of the properties outlined for the ideal PDT agent, jadomycins exhibit cytotoxic activity against both Gram positive and Gram negative bacteria as well as some drug-resistant cancer cell lines. This research program will determine whether direct DNA cleavage contributes to the overall cytotoxicity of the jadomycins at intracellular Cu(II) concentrations.  In addition, the DNA photocleaving properties of the jadomycins will be investigated, with particular attention on the mechanism that predominates with Cu(II) activation versus light activation.  The role that L-digitoxose, a structural feature of jadomycin B, plays in DNA cleavage will also be probed to better understand how glycone incorporation aids in DNA recognition.